The proposed project is to study the effects of interactions of parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and calcitonin (CT) on specific protein phosphorylation as an integral step in the intracellular events involved in the hormonal regulation of bone resorption. Phosphorylation of specific substrate proteins has been proposed as an important means of regulating responses to various cellular effectors, especially those that utilize cAMP as a second messenger. Both PTH, which stimulates bone resorption, and CT which inhibits basal na dstimulated resorption, stimulate cAMP production by bone. Since bone is a heterogeneous tissue, it is possible that PTH and CT act on different cell types in bone. 1,25-(OH)2D3, also directly stimulates bone resorption but has no effect alone on cAMP production, though it may affect the PTH response. We will utilize fetal rat limb bones and neonatal mouse calvaria in organ culture, and isolated bone cells to characterize the hormonal effects on protein phosphorylation. The result of labeling with 32P-ATP in a cell-free system after incubation of whole tissue or cells with hormones will be examined to monitor resorption and phosphorylation simultaneously. 32P labeling of whole tissue or cells before hormonal treatment and subsequent assay of the level of 32P incorporated into specific proteins also will be done to measure changes in the activity of the affected enzymes or substratae proteins as well as in their levels. Polyacrylamide gel electrophoresis and autoradiography will be used to analyze the observed changes. Although PTH and 1,25-(OH)2D3 have the same ultimate effect on bone, the initiating steps in their mechanisms of action are probably different since PTH is a peptide and 1,25-(OH)2D3 is a steriod hormone. Defining specific effects on protein phosphorylation should further our understanding of the mechanism of PTH action and could help define the point of convergence of the actions of PTH and 1,25-(OH)2D3. CT interactions with both hormones could delineate which phosphorylation effects are important to the hormonal control of bone resorption and also help identify the actual target cells in bone for each hormone. Understanding the control of normal mineral homeostasis by these hormones would help to identify possible mechanisms underlying bone disease and allow better therapeutic managememt and/or prevention of abnormalities of calcium balance.